We are studying the structure of the micronuclear genome of the hypotrichus ciliated protozoan Oxytricha and the common structural features of the DNA pieces wich comprise the macronuclear genome, which is excised from the total (micronuclear) genome. We are screening cloned micronuclear DNA fragments for those containing a junction(s) between macronuclear and non-macronuclear sequences, in collaboration with Drs. Marshal Swanton and David Prescott (University of Colorado). Simultaneously, we'll be isolating cloned macronuclear DNA pieces which correspond to the selected micronuclear DNA clones. This will allow: 1) comparison of macronuclear sequences before and after excision; 2) Structural analysis of the junction(s); 3) Isolation of putative site-specific DNA-binding proteins and nucleases presumably involved in excision. Macronuclear DNA pieces appear to sport specific nicks in sites related to the terminal (excision) sites. We are working to visualize these nicks directly and study them in detail with DNA enzymes (kinase, ligase, polymerases, etc). Finally, an affinity chromatography procedure is being developed with the lac repressor operator system as a model to allow the identification and isolation of site-specific DNA binding proteins.